Vector System

ABSTRACT

The present invention provides a vector system comprising a mutated post-transcriptional regulatory element. In particular, the present invention relates to a mutated WPRE sequence that can efficiently express nucleotides of interest in a retroviral vector system. The present invention also relates to methods of delivering and expressing nucleotides of interest to a target cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.10/408,456, filed on Apr. 7, 2003, which is a continuation-in-part ofInternational Application No. PCT/GB01/04433, filed on Oct. 5, 2001,which claims priority to British Application No. GB 0024550.6, filed onOct. 6, 2000.

This application makes reference to U.S. application Ser. No.10/008,610, filed on Nov. 8, 2001, which claims priority to U.S.Provisional Application Ser. No. 60/247,604, filed on Nov. 9, 2000.

This application also makes reference to U.S. application Ser. No.10/841,603, filed on May 7, 2004, which is a continuation-in-part ofInternational application No. PCT/GB03/064665, filed on Feb. 3,2003,which claims priority to British application Nos. GB 0202403.2,filed on Feb. 1, 2002, and GB 0212768.6, filed on May 31, 2002.

This application also makes reference to U.S. Pat. Nos. 6,312,682 and6,312,683, the contents of which are expressly incorporated herein byreference.

All of the foregoing applications, as well as all documents cited in theforegoing applications (“application documents”) and all documents citedor referenced in the application documents are incorporated herein byreference. Also, all documents cited in this application (“herein-citeddocuments”) and all documents cited or referenced in herein-citeddocuments are incorporated herein by reference. In addition, anymanufacturer's instructions or catalogues for any products cited ormentioned in each of the application documents or herein-cited documentsare incorporated by reference. Documents incorporated by reference intothis text or any teachings therein can be used in the practice of thisinvention. Documents incorporated by reference into this text are notadmitted to be prior art.

FIELD OF THE INVENTION

The present invention relates to a vector system comprising a mutatedpost-transcriptional regulatory element. In particular, the presentinvention relates to a mutated WPRE sequence that can efficientlyexpress nucleotides of interest in a retroviral vector system. Thepresent invention also relates to methods of delivering and expressingnucleotides of interest to a target cells.

BACKGROUND OF THE INVENTION

Retroviral vector systems, such as lentiviral vector systems, have beenproposed as a delivery system for, inter alia, the transfer of anucleotide of interest to one or more sites of interest. Indeed, theconcept of using viral vectors for gene therapy is well known (Verma andSomia (1997) Nature 389:239-242). Retrovirus genomes contain accessorygenes, such as a rev gene, a tat gene, a vif gene, a nef gene, a vprgene or an S2 gene. The deletion of such accessory genes, particularlywhen using retroviral vector systems in gene therapy, is highlyadvantageous. Firstly, it permits vectors to be produced without genesnormally associated with disease in retroviral (e.g. HIV) infections.Secondly, the deletion of accessory genes permits the vector to packagemore heterologous DNA. Thirdly, genes whose function is unknown such asdUTPase and S2, may be omitted, thus reducing the risk of causingundesirable effects.

We have previously taught, e.g. in WO98/17815, how to remove many of theaccessory genes. Further, in WO99/45126, we describe codon optimisationof the gag-pol sequence as a means of seeking to overcome the Rev/RRErequirement for export and to enhance RNA stability. However, the needremains to provide strategies for the provision of useful and safe viralvectors, and efficient means for their production.

WO 98/18934 involves gene transfer systems, such as retroviral vectors;and there are other documents that may involve retroviral vectors (See,e.g., Naldini et al., 1996 Science 272, 263; PCT/GB96/01230; Bowtell etal., 1988 J. Virol. 62, 2464; Correll et al., 1994 Blood 84, 1812;Emerman and Temin 1984 Cell 39, 459; Ghattas et al., 1991 Mol. Cell.Biol. 11, 5848; Hantzopoulos et al., 1989 PNAS 86, 3519; Hatzoglou etal., 1991 J. Biol. Chem 266, 8416; Hatzoglou et al., 1988 J. Biol. Chem263, 17798; Li et al., 1992 Hum. Gen. Ther. 3, 381; McLachlin et al.,1993 Virol. 195, 1; Overell et al., 1988 Mol. Cell Biol. 8, 1803;Scharfman et al., 1991 PNAS 88, 4626; Vile et al., 1994 Gene Ther 1,307; Xu et al., 1989 Virol. 171, 331; Yee et al., 1987 PNAS 84, 5197;WO99/15683; Verma and Somia (1997) Nature 389:239-242; page 446, Chapter9 of Coffin et al “Retroviruses” 1997 Cold Spring Harbour LaboratoryPress).

Post-Transcriptional Regulatory Elements

One shortcoming of retroviral vectors, whether based on retroviruses orlentiviruses, is their frequent inability to generate high levels ofgene expression, particularly in vivo. Many steps, both transcriptionaland post-transcriptional, are involved in regulating gene expression.Therefore, it is possible to enhance expression of transgenes deliveredby retroviral vectors through the addition of elements known topost-transcriptionally increase gene expression. The best-known exampleis the inclusion of introns within the expression cassette (Choi, T. etal, (1991) Mol. Cell. Biol. 9: 3070-3074). Many gene transferexperiments, performed both in vitro and in vivo, have demonstrated thatthe presence of an intron can facilitate gene expression.

Other types of elements can also be used to stimulate heterologous geneexpression post-transcriptionally. These elements, unlike introns, areadvantageous in that they do not require splicing events. For instance,previous studies have suggested that the hepatitis B virus (HBV)post-transcriptional regulatory element (PRE) and an intron arefunctionally equivalent (Huang, Z. M. and Yen, T. S. (1995) Mol. Cell.Biol. 15: 3864-3869). Woodchuck hepatitis virus (WHV), a close relativeof HBV, also harbors a PRE (hereinafter referred to as WPRE; see U.S.Pat. Nos. 6,136,597 and 6,287,814). The WPRE has been shown to besignificantly more active than its HBV counterpart, correlating to thepresence of additional cis-acting sequences not found in the HBV PRE.Insertion of the WPRE in lentiviral vectors resulted in significantstimulation of expression of reporter genes such as luciferase and greenfluorescent protein (GFP) in a variety of cells spanning differentspecies (Zufferey, R. et al, (1999) J. Virol 73: 2886-2892). Stimulationwas irrespective of the cycling status of transduced cells.

The WPRE contains three cis-acting sequences important for its functionin enhancing expression levels. However, in addition, it contains afragment of approximately 180 base pairs (bp), comprising the 5′ end ofthe WHV X protein open reading frame, together with its associatedpromoter. The full-length X protein has been implicated in tumorigenesis(Flajolet, M. et al, (1998) J. Virol. 72: 6175-6180). Cis-activation ofmyc family oncogenes due to the insertion of viral DNA into the hostgenome is known to be a key mechanism of WHV-mediated carcinogenesis(Buendia, M. A. (1994) In C. Bréchot (ed.), Primary liver cancer:etiological and progression factors, p. 211-224; CRC Press, Boca Raton,Fla.; Fourel, G. (1994) In F. Tronche and M. Yaniv (ed.), Liver geneexpression, p. 297-343; R.G. Landes Company, Austin, Tex.).

The present inventors have now shown that mutation of a region of theWPRE corresponding to the X protein ORF ablates the tumorigenic activityof the X protein, thereby allowing the WPRE to be used safely inretroviral and lentiviral expression vectors to enhance expressionlevels of heterologous genes or nucleotides of interest. Moreover, themodified WPRE can be used to identify genes involved in tumorigenesis byidentifying its integration site in the chromosomal DNA of cells ofinterest.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention, there is providedan isolated nucleic acid molecule comprising a woodchuckpost-transcriptional regulatory element (WPRE) containing an X region,wherein the WPRE has a mutation in the X region, whereby expression of afunctional X protein is prevented. In a preferred embodiment, theisolated nucleic acid molecule comprises the sequence of SEQ ID NO:1.

In a preferred embodiment, the X region comprises a sufficient number ofbase pair changes such that reversion to the wild type WPRE sequence issubstantially prevented. In particularly preferred embodiments, the Xregion sequence comprises at least six, seven, eight, nine, ten, eleven,twelve or thirteen base pair changes, relative to the wild type WPREsequence. Preferably, the X region of the invention comprises thesequence:

    5    10   15 GTCTGCTGAGAGACTCGG (SEQ ID NO:2)

Preferably, the X region comprises a promoter sequence and the mutationis partly or entirely in the promoter sequence. Preferably, the wildtype WPRE X region promoter sequence comprises the sequence:

    5    10   15   20 GGGGAAGCTGACGTCCTTTCC (SEQ ID NO:3)

Preferably, the X region promoter sequence comprises at least six,seven, eight, nine, ten or eleven base pair changes relative to the wildtype WPRE sequence. Even more preferably, the X region promoter sequencehas a mutation at one or more of positions 12, 13, 15, 16, 17, 18, 19and/or 20 of the wild type sequence. Advantageously, the X regionpromoter sequence of the invention is:

    5    10   15   20 GGGAAGGTCTGCTGAGACTC (SEQ ID NO:4)

Alternatively, or in addition, the X region comprises an initiationcodon and the mutation is partly or entirely in the initiation codon.Preferably, the initiation codon of the wild type WPRE is A at position1, T at position 2 and G at position 3. In one embodiment, theinitiation codon comprises a nucleotide other than T at position 2. Inanother embodiment, the mutation comprises at least two base pairchanges relative to the wild type WPRE sequence. Preferably, the Xregion initiation codon is GGG.

Preferably, the X protein is not expressed or is non-functional.

In a second aspect of the present invention, a retroviral vector genomecomprising at least one NOI and the isolated nucleic acid moleculeaccording to the first aspect of the invention is provided.

Preferably, the retroviral vector genome is a lentiviral vector genome.Particularly, the lentiviral vector genome can be a minimal lentiviralvector genome. The lentiviral vector genome can be derived from a viralspecies selected from the group consisting of human immunodeficiencyvirus (HIV), simian immunodeficiency virus (SIV), visna/maedi virus(VMV), caprine arthritis-encephalitis virus (CAEV), equine infectiousanaemia virus (EIAV), feline immunodeficiency virus (FIV), and bovineimmunodeficiency virus (BIV). Preferably, the lentiviral vector genomeis derived from a non-primate lentivirus.

In another preferred embodiment, a nucleic acid sequence encoding Rev,or a functional equivalent thereof, is disrupted such that the nucleicacid sequence is incapable of encoding the functional Rev or is removedfrom the vector genome.

In yet another preferred embodiment, a nucleic acid sequence encodingTat is disrupted such that the nucleic acid sequence is incapable ofencoding functional Tat or is removed from the vector genome.

A preferred embodiment of the present invention provides a retroviralvector genome that comprises a central polypurine tract (cPPT) sequence.In another preferred embodiment, the retroviral vector genome comprisesa gag-packaging signal having ATG motifs, and wherein the ATG motifs areATTG motifs.

In another preferred embodiment, the retroviral vector genome ismulticistronic and can comprise at least one internal regulatoryelement. Preferably, the internal regulatory element is a promoter or aninternal ribosomal entry site (IRES).

According to a third aspect of the present invention, there is provideda retroviral vector system for producing a retrovirus-derived vectorparticle, comprising (i) the retroviral vector genome according to thesecond aspect of the invention, (ii) a nucleotide sequence encodingretroviral gag and pol proteins; (iii) nucleotide sequences encodingother essential viral packaging components not encoded by the nucleotidesequence of (ii). Preferably, nucleic acid sequence(s) encoding at leastone of Vpr, Vif, Tat, Nef, or analogous auxiliary genes, from theretrovirus from which the particles are derived, are disrupted such assaid nucleic acid sequence(s) are incapable of encoding functional Vpr,Vif, Tat, Nef, or analogous auxiliary proteins, or are removed from thesystem.

Preferably, the vector system is pseudotyped with at least part of aheterologous env protein. In particular, the heterologous env protein isderivable from Rabies-G or VSV-G.

In a fifth aspect of the invention, a viral particle produced from theretroviral vector system of the present invention is provided.

A sixth aspect of the invention provides a cell that has been transducedwith the retroviral vector system of the present invention.

In a seventh aspect of the invention, a composition comprising theretroviral vector genome of the present invention, together with acarrier or a diluent, is provided.

An eighth aspect of the invention provides a composition comprising aviral particle of the present invention, together with a carrier or adiluent.

A ninth aspect of the invention provides a method of delivering at leastone NOI to a target cells, comprising introducing the retroviral vectorgenome of the present invention into the target cell, whereby the NOI isdelivered to the target cell.

According to a tenth aspect, there is provided a method of identifyinggenes involved in tumorigenesis, comprising the steps of introducing theisolated nucleic acid molecule of the present invention into a cell ofinterest, whereby the nucleic acid is recombined into chromosomal DNA ofthe cell of interest; determining whether the cell of interest forms atumor; and, if the cell of interest forms a tumor, locating a site ofrecombination in the chromosomal DNA, and identifying a gene near oradjacent to the site of recombination; thereby identifying the geneinvolved in tumorigenesis.

The present invention will now be described only by way of example, inwhich reference will be made to the following Figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of EIAV minimal vectors.

FIG. 2 shows a plasmid map of pONY8G

FIG. 3 shows a plasmid map of pONY8.1G

FIG. 4 shows a plasmid map of pONY8Z

FIG. 5 shows the effect of mutations within the WPRE on the expressionlevel of a reporter gene, GFP. FACS analysis was used to calculate theMFI of eGFP expression in D17 cells that were between 1% and 10%GFP-positive. The MFI±SD of eGFP expression in pSMART3G WPRE andpSMART3G WPREMut is shown.

FIG. 6 shows a comparison of the nucleotide sequence of wild-type WPREsequence from WHV8 (WT) (SEQ ID NO:5), WPRE from SMART2 vectors (SMT)(SEQ ID NO:6), and WPRE incorporating nucleotide changes designed toprevent transcription from the X promoter and translation from the Xprotein initiation codon (MUT) (SEQ ID NO:1). The region containing theX promoter and the X protein initiation codon is underlined. Differencesbetween the WPRE nucleotide sequences are shown in bold type.

FIG. 7 shows a plasmid map of pONY8.9.4 MV opti Y. This vector comprisesa modified form of WPRE, at nucleotides 8206-8795 of the vector plasmid.

FIGS. 8A-8H show the sequence of pONY8.9.4 MV opti Y (SEQ ID NO:7).

DETAILED DESCRIPTION OF THE INVENTION

Various preferred features and embodiments of the present invention willnow be described by way of non-limiting example. Although in general,the techniques mentioned herein are well known in the art, reference maybe made in particular to Sambrook, et al., Molecular Cloning, ALaboratory Manual (1989) and Ausubel et al., Short Protocols inMolecular Biology (1999) 4^(th) Ed., John Wiley & Sons, Inc. (as well asthe complete version of Current Protocols in Molecular Biology).

As used herein, the term “operably linked” means that the componentsdescribed are in a relationship permitting them to function in theirintended manner.

Post-Transcriptional Regulatory Elements

The Woodchuck hepatitis virus (WHV) post-transcriptional regulatoryelement (WPRE) can enhance expression from a number of different vectortypes including lentiviral vectors (U.S. Pat. Nos. 6,136,597; 6,287,814;Zufferey, R., et al. (1999) J. Virol. 73: 2886-92). Without wanting tobe bound by theory, this enhancement is thought to be due to improvedRNA processing at the post-transcriptional level, resulting in increasedlevels of nuclear transcripts. A two-fold increase in mRNA stabilityalso contributes to this enhancement (Zufferey, R., et al. ibid). Thelevel of enhancement of protein expression from transcripts containingthe WPRE versus those without the WPRE has been reported to be around2-to-5 fold, and correlates well with the increase in transcript levels.This has been demonstrated with a number of different transgenes(Zufferey, R., et al. ibid).

The WPRE contains three cis-acting sequences important for its functionin enhancing expression levels. In addition, it contains a fragment ofapproximately 180 bp comprising the 5′-end of the WHV X protein ORF(full length ORF is 425 bp), together with its associated promoter.Translation from transcripts initiated from the X promoter results information of a protein representing the NH₂-terminal 60 amino acids ofthe X protein. This truncated X protein can promote tumorigenesis,particularly if the truncated X protein sequence is integrated into thehost cell genome at specific loci (Balsano, C. et al, (1991) Biochem.Biophys Res. Commun. 176: 985-92; Flajolet, M. et al, (1998) J. Virol.72: 6175-80; Zheng, Y. W., et al, (1994) J. Biol. Chem. 269: 22593-8;Runkel, L., et al, (1993) Virology 197: 529-36). Therefore, expressionof the truncated X protein could promote tumorigenesis if delivered tocells of interest, precluding safe use of wild-type WPRE sequences.

As used herein, the “X region” of the WPRE is defined as comprising atleast the first 60-amino acids of the X protein ORF, including thetranslation initiation codon, and its associated promoter. A“functional” X protein is defined herein as a truncated X protein thatis capable of promoting tumorigenesis, or a transformed phenotype, whenexpressed in cells of interest. A “non-functional” X protein in thecontext of this application is defined as an X protein that is incapableof promoting tumorigenesis in cells of interest.

The present inventors have introduced mutations into the WPRE sequence,and found that the mutations prevent expression of a functional Xprotein, thereby preventing tumorigenesis in the cells of interest.Preferably, these mutations are introduced into the promoter region ofthe X protein, or into the translation initiation codon of the Xprotein. The present inventors have found that the nature of thesemutations, in addition to preventing expression of a functional Xprotein, also prevents the reversion back to the wild-type WPREsequences, which can be accomplished by the low-fidelity proofreadingactivity of viral-encoded reverse transcriptase present in the vectorgenomes of the present invention.

A “mutation” can comprise one or more amino acid deletions, additions,or substitutions.

Accordingly, one aspect of the present invention provides an isolatednucleic acid molecule comprising a WPRE containing an X region, whereinthe WPRE has a mutation in the X region whereby expression of afunctional X protein is prevented. In preferred embodiments, themutation is in the promoter sequence of the X region or in theinitiation codon of the X region. Preferably, as a result of thesemutations, the X protein is not expressed, or is non-functional.

Liver tumours can occur sporadically when vectors expressing thewild-type WPRE are transduced in utero. Despite the presence of thewild-type WPRE, tumours do not occur in the case of every integrationevent. Therefore, other secondary factors are presumably required topromote tumorigenesis. These secondary factors can include those arisingfrom insertional mutagenesis as a result of integration of the viralvector genome comprising the wild-type WPRE. As a consequence,expression of a known factor that is involved in, but not sufficientfor, tumorigenesis (for example, a mutated tumour suppressor gene) inthe context of an integrating vector can lead to integration within, oradjacent to, a secondary gene that has a role in tumour generation andtogether with expression of the known factor, can lead to tumorigenesis.The tumour mass can then be removed and its genomic DNA isolated. Usingthis material, the integration site can be located and the unknownsecondary factor can be identified. Location of the integration site canbe achieved by standard methods known to the skilled artisan such as,but not limited to, Southern hybridisation and genomic PCR.

Therefore, in an embodiment of the present invention, methods foridentifying a gene involved in tumorigenesis is provided, comprising thesteps of introducing the isolated nucleic acid according to the firstaspect of the invention, into a cell of interest, whereby the nucleicacid is recombined into chromosomal DNA of the cell of interest,determining whether the cell of interest forms a tumor; and, if the cellof interest forms a tumor: locating a site of recombination in thechromosomal DNA, and identifying a gene near or adjacent to the site ofrecombination, thereby identifying the gene involved in tumorigenesis.

Retroviruses

The concept of using viral vectors for gene therapy is well known (Vermaand Somia (1997) Nature 389:239-242).

There are many retroviruses. For the present application, the term“retrovirus” includes: murine leukemia virus (MLV), humanimmunodeficiency virus (HIV), equine infectious anaemia virus (EIAV),mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinamisarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murineosteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV),Abelson murine leukemia virus (A-MLV), Avian myelocytomatosis virus-29(MC29), and Avian erythroblastosis virus (AEV) and all otherretroviridiae including lentiviruses.

A detailed list of retroviruses may be found in Coffin et al(“Retroviruses” 1997 Cold Spring Harbour Laboratory Press Eds: J MCoffin, S M Hughes, H E Varmus pp 758-763).

Lentiviruses also belong to the retrovirus family, but they can infectboth dividing and non-dividing cells (Lewis et al (1992) EMBO J.3053-3058).

The lentivirus group can be split into “primate” and “non-primate”.Examples of primate lentiviruses include the human immunodeficiencyvirus (HIV), the causative agent of human acquired immunodeficiencysyndrome (AIDS), and the simian immunodeficiency virus (SIV). Thenon-primate lentiviral group includes the prototype “slow virus”visna/maedi virus (VMV), as well as the related caprinearthritis-encephalitis virus (CAEV), equine infectious anaemia virus(EIAV) and the more recently described feline immunodeficiency virus(FIV) and bovine immunodeficiency virus (BIV).

Details on the genomic structure of some lentiviruses may be found inthe art. By way of example, details on HIV and EIAV may be found fromthe NCBI Genbank database (i.e. Genome Accession Nos. AF033819 andAF033820 respectively). Details of HIV variants may also be found in theHIV databases maintained by Los Alamos National Laboratory. Details ofEIAV clones may be found at the NCBI database maintained by the NationalInstitutes of Health.

During the process of infection, a retrovirus initially attaches to aspecific cell surface receptor. On entry into the susceptible host cell,the retroviral RNA genome is then copied to DNA by the virally encodedreverse transcriptase, which is carried inside the parent virus. ThisDNA is transported to the host cell nucleus where it subsequentlyintegrates into the host genome. At this stage, it is typically referredto as the provirus. The provirus is stable in the host chromosome duringcell division and is transcribed like other cellular genes. The provirusencodes the proteins and other factors required to make more virus,which can leave the cell by a process sometimes called “budding”.

Each retroviral genome comprises genes called gag, pol and env, whichcode for virion proteins and enzymes. These genes are flanked at bothends by regions called long terminal repeats (LTRs). The LTRs areresponsible for proviral integration, and transcription. They also serveas enhancer-promoter sequences. In other words, the LTRs can control theexpression of the viral genes. Encapsidation of the retroviral RNAsoccurs by virtue of a psi sequence located at the 5′ end of the viralgenome.

The LTRs themselves are identical sequences that can be divided intothree elements, which are called U3, R and U5. U3 is derived from thesequence unique to the 3′ end of the RNA. R is derived from a sequencerepeated at both ends of the RNA and U5 is derived from the sequenceunique to the 5′end of the RNA. The sizes of the three elements can varyconsiderably among different retroviruses.

For the viral genome, the site of transcription initiation is at theboundary between U3 and R in the left hand side LTR and the site of poly(A) addition (termination) is at the boundary between R and U5 in theright hand side LTR. U3 contains most of the transcriptional controlelements of the provirus, which include the promoter and multipleenhancer sequences responsive to cellular and in some cases, viraltranscriptional activator proteins. Some retroviruses have any one ormore of the following genes that code for proteins that are involved inthe regulation of gene expression: tat, rev, tax and rex.

With regard to the structural genes gag, pol and env themselves; gagencodes the internal structural protein of the virus. Gag protein isproteolytically processed into the mature proteins MA (matrix), CA(capsid) and NC (nucleocapsid). The pol gene encodes the reversetranscriptase (RT), which contains DNA polymerase, associated RNase Hand integrase (IN), which mediate replication of the genome. The envgene encodes the surface (SU) glycoprotein and the transmembrane (TM)protein of the virion, which form a complex that interacts specificallywith cellular receptor proteins. This interaction leads ultimately toinfection by fusion of the viral membrane with the cell membrane.

Retroviruses may also contain “additional” genes, which code forproteins other than gag, pol and env. Examples of additional genesinclude in HIV, one or more of vif, vpr, vpx, vpu, tat, rev and nef EIAVhas, for example, the additional genes S2 and dUTPase.

Proteins encoded by additional genes serve various functions, some ofwhich may be duplicative of a function provided by a cellular protein.In EIAV, for example, tat acts as a transcriptional activator of theviral LTR. It binds to a stable, stem-loop RNA secondary structurereferred to as TAR. Rev regulates and co-ordinates the expression ofviral genes through rev-response elements (RRE). The mechanisms ofaction of these two proteins are thought to be broadly similar to theanalogous mechanisms in the primate viruses. The function of S2 isunknown. In addition, an EIAV protein, Ttm, has been identified that isencoded by the first exon of tat spliced to the env coding sequence atthe start of the transmembrane protein.

Delivery Systems

Retroviral vector systems have been proposed as a delivery system for,inter alia, the transfer of a NOI to one or more sites of interest. Thetransfer can occur in vitro, ex vivo, in vivo, or combinations thereof.Retroviral vector systems have even been exploited to study variousaspects of the retrovirus life cycle, including receptor usage, reversetranscription and RNA packaging (reviewed by Miller, 1992 Curr TopMicrobiol Immunol 158:1-24).

A recombinant retroviral vector particle is capable of transducing arecipient cell with an NOI. Once within the cell, the RNA genome fromthe vector particle is reverse transcribed into DNA and integrated intothe DNA of the recipient cell.

As used herein, the term “vector genome” refers to the RNA constructpresent in the retroviral vector particle and/or the integrated DNAconstruct. The term also embraces a separate or isolated DNA constructcapable of encoding such an RNA genome. A retroviral or lentiviralgenome should comprise at least one component part derivable from aretrovirus or a lentivirus. The term “derivable” is used in its normalsense as meaning a nucleotide sequence or a part thereof, which need notnecessarily be obtained from a virus such as a lentivirus but insteadcould be derived therefrom. By way of example, the sequence may beprepared synthetically or by use of recombinant DNA techniques.Preferably the genome comprises a psi region (or an analogous componentthat is capable of causing encapsidation).

The viral vector genome is preferably “replication defective”, by whichwe mean that the genome does not comprise sufficient genetic informationalone to enable independent replication to produce infectious viralparticles within the recipient cell. In a preferred embodiment, thegenome lacks a functional env, gag or pol gene.

The viral vector genome may comprise some or all of the long terminalrepeats (LTRs). Preferably the genome comprises at least part of theLTRs or an analogous sequence, which is capable of mediating proviralintegration, and transcription. The sequence may also comprise or act asan enhancer-promoter sequence.

The viral vector genome of the second aspect of the invention may beprovided as a kit of parts. For example, the kit may comprise (i) aplasmid or plasmids containing the NOIs and internal regulatorysequences, such as, for example, a promoter or an IRES sequence(s); and(ii) a retroviral genome construct with suitable restriction enzymerecognition sites for cloning the NOIs and internal regulatorysequence(s) into the viral genome.

It is known that the separate expression of the components required toproduce a retroviral vector particle on separate DNA sequencescointroduced into the same cell will yield retroviral particles carryingdefective retroviral genomes that carry therapeutic genes (e.g. Reviewedby Miller 1992). This cell is referred to as the producer cell (seebelow).

There are two common procedures for generating producer cells. In one,the sequences encoding retroviral Gag, Pol and Env proteins areintroduced into the cell and stably integrated into the cell genome; astable cell line is produced which is referred to as the packaging cellline. The packaging cell line produces the proteins required forpackaging retroviral RNA but it cannot bring about encapsidation due tothe lack of a psi region. However, when a vector genome having a psiregion is introduced into the packaging cell line, the helper proteinscan package the psi-positive recombinant vector RNA to produce therecombinant virus stock. This can be used to transduce the NOI intorecipient cells. The recombinant virus whose genome lacks all genesrequired to make viral proteins can infect only once and cannotpropagate. Hence, the NOI is introduced into the host cell genomewithout the generation of potentially harmful retrovirus. A summary ofthe available packaging lines is presented in “Retroviruses” (1997 ColdSpring Harbour Laboratory Press Eds: J M Coffin, S M Hughes, H E Varmuspp 449).

The present invention also provides a packaging cell line comprising aviral vector genome of the present invention. For example, the packagingcell line may be transduced with a viral vector system comprising thegenome or transfected with a plasmid carrying a DNA construct capable ofencoding the RNA genome. The present invention also provides aretroviral (or lentiviral) vector particle produced by such a cell.

The second approach is to introduce the three different DNA sequencesthat are required to produce a retroviral vector particle i.e. the envcoding sequences, the gag-pol coding sequence and the defectiveretroviral genome containing one or more NOIs into the cell at the sametime by transient transfection and the procedure is referred to astransient triple transfection (Landau & Littman 1992; Pear et al 1993).The triple transfection procedure has been optimised (Soneoka et al1995; Finer et al 1994). WO 94/29438 describes the production ofproducer cells in vitro using this multiple DNA transient transfectionmethod.

The components of the viral system, which are required to complement thevector genome, may be present on one or more “producer plasmids” fortransfecting into cells.

The present invention also provides a vector system for producing aretrovirus-derived particle, comprising

-   -   (i) a retroviral genome according to the second aspect of the        invention;    -   (ii) a nucleotide sequence coding for retroviral gag and pol        proteins;    -   (iii) nucleotide sequences encoding other essential viral        packaging components not encoded by the nucleotide sequence of        (ii).

Preferably, the nucleic acid sequence(s) encoding at least one of Vpr,Vif, Tat, Nef, or analogous auxiliary genes, from the retrovirus fromwhich the particles are derived, are disrupted such as said nucleic acidsequence(s) are incapable of encoding functional Vpr, Vif, Tat, Nef, oranalogous auxiliary proteins, or are removed from the system.

The present invention also provides a cell transfected with such avector system and a retroviral vector particle produced by such a cell.Preferably the gag-pol sequence is codon optimised for use in theparticular producer cell (see below).

The env protein encoded by the nucleotide sequence of iii) may be ahomologous retroviral or lentiviral env protein. Alternatively, it maybe a heterologous env, or an env from a non-retro or lentivirus (seebelow under “pseudotyping”).

The term “viral vector system” is used generally to mean a kit of partsthat can be used when combined with other necessary components for viralparticle production to produce viral particles in host cells. Forexample, the retroviral vector genome may lack one or more of the genesneeded for viral replication. This may be combined in a kit with afurther complementary nucleotide sequence or sequences, for example onone or more producer plasmids. By cotransfection of the genome togetherwith the producer plasmid(s), the necessary components should beprovided for the production of infectious viral particles.

Alternatively, the complementary nucleotide sequence(s) may be stablypresent within a packaging cell line that is included in the kit.

The present invention also relates to a retroviral vector system, whichis capable of delivering an RNA genome to a recipient cell, wherein thegenome is longer than the wild type genome of the lentivirus. The vectorsystem may, for example, be an EIAV vector system.

Preferably the RNA genome of the vector system has up to 5%, morepreferably up to 10% or even up to 30% more bases than the wild-typegenome. Preferably the RNA genome is about 10% longer than the wild-typegenome. For example, wild type EIAV comprises an RNA genome ofapproximately 8 kb. An EIAV vector system of the present invention mayhave an RNA genome of up to (preferably about) 8.8 kb.

Preferably the retroviral vector system of the present invention is aself-inactivating (SIN) vector system.

By way of example, self-inactivating retroviral vector systems have beenconstructed by deleting the transcriptional enhancers or the enhancersand promoter in the U3 region of the 3′ LTR. After a round of vectorreverse transcription and integration, these changes are copied intoboth the 5′ and the 3′ LTRs, producing a transcriptionally inactiveprovirus. However, any promoter(s) internal to the LTRs in such vectorswill still be transcriptionally active. This strategy has been employedto eliminate effects of the enhancers and promoters in the viral LTRs ontranscription from internally placed genes. Such effects includeincreased transcription or suppression of transcription. This strategycan also be used to eliminate downstream transcription from the 3′ LTRinto genomic DNA. This is of particular concern in human gene therapywhere it may be important to prevent the adventitious activation of anendogenous oncogene (Yu et al., (1986) PNAS 83: 3194-98; Marty et al.,(1990) Biochimie 72: 885-7; Naviaux et al., (1996) J. Virol. 70: 5701-5;Iwakuma et al., (1999) Virol. 261: 120-32; Deglon et al., (2000) HumanGene Therapy 11: 179-90).

Preferably a recombinase-assisted mechanism is used, which facilitatesthe production of high titre regulated lentiviral vectors from theproducer cells of the present invention.

As used herein, the term “recombinase assisted system” includes, but isnot limited to, a system using the Cre recombinase/1oxP recognitionsites of bacteriophage P1 or the site-specific FLP recombinase of S.cerevisiae, which catalyses recombination events between 34 bp FLPrecognition targets (FRTs).

The site-specific FLP recombinase of S. cerevisiae, which catalysesrecombination events between 34 bp FLP recognition targets (FRTs), hasbeen configured into DNA constructs to generate high level producer celllines using recombinase-assisted recombination events (Karreman et al(1996) NAR 24:1616-1624). A similar system has been developed using theCre recombinase/1oxP recognition sites of bacteriophage P1 (Vanin et al(1997) J. Virol 71:7820-7826). This was configured into a lentiviralgenome such that high titre lentiviral producer cell lines weregenerated.

By using producer/packaging cell lines, it is possible to propagate andisolate quantities of retroviral vector particles (e.g. to preparesuitable titres of the retroviral vector particles) for subsequenttransduction of, for example, a site of interest (such as adult braintissue). Producer cell lines are usually better for large-scaleproduction or vector particles.

Transient transfection has numerous advantages over the packaging cellmethod. In this regard, transient transfection avoids the longer timerequired to generate stable vector-producing cell lines and is used ifthe vector genome or retroviral packaging components are toxic to cells.If the vector genome encodes toxic genes or genes that interfere withthe replication of the host cell, such as inhibitors of the cell cycleor genes that induce apoptosis, it may be difficult to generate stablevector-producing cell lines, but transient transfection can be used toproduce the vector before the cells die. Also, cell lines have beendeveloped using transient infection that produce vector titre levelsthat are comparable to the levels obtained from stable vector-producingcell lines (Pear et al 1993, PNAS 90:8392-8396).

Producer cells/packaging cells can be of any suitable cell type.Producer cells are generally mammalian cells, but can be, for example,insect cells.

As used herein, the term “producer cell” or “vector producing cell”refers to a cell that contains all the elements necessary for productionof retroviral vector particles.

Preferably, the producer cell is obtainable from a stable producer cellline.

Preferably, the producer cell is obtainable from a derived stableproducer cell line.

Preferably, the producer cell is obtainable from a derived producer cellline.

As used herein, the term “derived producer cell line” is a transducedproducer cell line that has been screened and selected for highexpression of a marker gene. Such cell lines support high-levelexpression from the retroviral genome. The term “derived producer cellline” is used interchangeably with the term “derived stable producercell line” and the term “stable producer cell line.

Preferably the derived producer cell line includes, but is not limitedto, a retroviral and/or a lentiviral producer cell.

Preferably the derived producer cell line is an HIV or EIAV producercell line, more preferably an EIAV producer cell line.

Preferably the envelope protein sequences, and nucleocapsid sequencesare all stably integrated in the producer and/or packaging cell.However, one or more of these sequences could also exist in episomalform and gene expression could occur from the episome.

As used herein, the term “packaging cell” refers to a cell that containsthose elements necessary for production of infectious recombinant virusthat are lacking in the RNA genome. Typically, such packaging cellscontain one or more producer plasmids, which are capable of expressingviral structural proteins (such as codon optimised gag-pol and env) butthey do not contain a packaging signal.

The term “packaging signal” which is referred to interchangeably as“packaging sequence” or “psi” is used in reference to the non-coding,cis-acting sequence required for encapsidation of retroviral RNA strandsduring viral particle formation. In HIV-1, this sequence has been mappedto loci extending from upstream of the major splice donor site (SD) toat least the gag start codon.

Packaging cell lines suitable for use with the above-described vectorconstructs may be readily prepared (see also WO 92/05266), and utilisedto create producer cell lines for the production of retroviral vectorparticles. As already mentioned, a summary of the available packaginglines is presented in “Retroviruses” (as above).

Also as discussed above, simple packaging cell lines, comprising aprovirus in which the packaging signal has been deleted, have been foundto lead to the rapid production of undesirable replication competentviruses through recombination. In order to improve safety,second-generation cell lines have been produced, wherein the 3 ′LTR ofthe provirus is deleted. In such cells, two recombinations would benecessary to produce a wild type virus. A further improvement involvesthe introduction of the gag-pol genes and the env gene on separateconstructs so-called third generation packaging cell lines.

These constructs are introduced sequentially to prevent recombinationduring transfection.

Preferably, the packaging cell lines are second-generation packagingcell lines.

Preferably, the packaging cell lines are third generation packaging celllines.

In these split-construct, third generation cell lines, a furtherreduction in recombination may be achieved by changing the codons. Thistechnique, based on the redundancy of the genetic code, aims to reducehomology between the separate constructs, for example, between theregions of overlap in the gag-pol and env open reading frames.

The packaging cell lines are useful for providing the gene productsnecessary to encapsidate and provide a membrane protein for a high titrevector particle production. The packaging cell may be a cell cultured invitro, such as a tissue culture cell line. Suitable cell lines include,but are not limited to, mammalian cells, such as murine fibroblastderived cell lines or human cell lines. Preferably the packaging cellline is a primate or human cell line, such as for example: HEK293,293-T, TE671, HT1080.

It is highly desirable to use high-titre virus preparations in bothexperimental and practical applications. Techniques for increasing viraltitre include using a psi plus packaging signal as discussed above andconcentration of viral stocks.

As used herein, the term “high titre” means an effective amount of aretroviral vector or particle that is capable of transducing a targetsite such as a cell.

As used herein, the term “effective amount” means an amount of aretroviral or lentiviral vector or vector particle that is sufficient toinduce expression of the NOIs at a target site.

A high-titre viral preparation for a producer/packaging cell is usuallyon the order of 10⁵ to 10⁷ &retrovirus particles per ml. Fortransduction in tissues such as the brain, it is necessary to use verysmall volumes, so the viral preparation is concentrated byultracentrifugation. The resulting preparation should have at least 10⁸t.u./ml, preferably from 10⁸ to 10⁹ t.u./ml, more preferably at least10⁹ t.u./ml. (The titer is expressed in transducing units per ml(t.u./ml) as titred on a standard D17 cell line). Other methods ofconcentration such as ultrafiltration or binding to and elution from amatrix may be used.

The expression products encoded by the NOIs may be proteins that aresecreted from the cell. Alternatively, the NOI expression products arenot secreted and are active within the cell. For some applications, itis preferred for the NOI expression product to demonstrate a bystandereffect or a distant bystander effect; that is the production of theexpression product in one cell leading to the modulation of additional,related cells, either neighbouring or distant (e.g. metastatic), whichpossess a common phenotype. Zennou et al., (2000) Cell 101: 173;Folleuzi et al., (2000) Nat. Genetics 25: 217; Zennou et al., (2001)Nat. Biotechnol. 19: 446.

The presence of a sequence termed the central polypurine tract (cPPT)may improve the efficiency of gene delivery to non-dividing cells. Thiscis-acting element is located, for example, in the EIAV polymerasecoding region element. Preferably the genome of the present inventioncomprises a cPPT sequence.

In addition, the viral genome may comprise a translational enhancer.

The NOIs may be operatively linked to one or more promoter/enhancerelements. Transcription of one or more NOIs may be under the control ofviral LTRs or alternatively promoter-enhancer elements. Preferably thepromoter is a strong viral promoter such as CMV, or is a cellularconstitutive promoter such as PGK, beta-actin or EF1 alpha. The promotermay be regulated or tissue-specific. The control of expression can alsobe achieved by using such systems as the tetracycline system thatswitches gene expression on or off in response to outside agents (inthis case tetracycline or its analogues).

Pseudotyping

In the design of retroviral vector systems, it is desirable to engineerparticles with different target cell specificities to the native virus,to enable the delivery of genetic material to an expanded or alteredrange of cell types. One manner in which to achieve this is byengineering the virus envelope protein to alter its specificity. Anotherapproach is to introduce a heterologous envelope protein into the vectorparticle to replace or add to the native envelope protein of the virus.

The term pseudotyping means incorporating in at least a part of, orsubstituting a part of, or replacing all of, an env gene of a viralgenome with a heterologous env gene, for example, an env gene fromanother virus. Pseudotyping is not a new phenomenon and examples may befound in WO 99/61639, WO-A-98/05759, WO-A-98/05754, WO-A-97/17457,WO-A-96/09400, WO-A-91/00047 and Mebatsion et al 1997 Cell 90, 841-847.

In a preferred embodiment of the present invention, the vector system ispseudotyped with a gene encoding at least part of the rabies G protein.Examples of rabies G pseudotyped retroviral vectors may be found inWO99/61639. In a further preferred embodiment of the present invention,the vector system is pseudotyped with a gene encoding at least part ofthe VSV-G protein. Examples of VSV-G pseudotyped retroviral vectors maybe found in U.S. Pat. No. 5,817,491.

It has been demonstrated that a retrovirus or lentivirus minimal systemcan be constructed from HIV, SIV, FIV, and EIAV viruses. Such a systemrequires none of the additional genes vif vpr, vpx, vpu, tat, rev andnef for either vector production or for transduction of dividing andnon-dividing cells. It has also been demonstrated that an EIAV minimalvector system can be constructed which does not require S2 for eithervector production or for transduction of dividing and non-dividingcells. The deletion of additional genes is highly advantageous. Firstly,it permits vectors to be produced without the genes associated withdisease in lentiviral (e.g. HIV) infections. In particular, tat isassociated with disease. Secondly, the deletion of additional genespermits the vector to package more heterologous DNA. Thirdly, geneswhose function is unknown, such as S2, may be omitted, thus reducing therisk of causing undesired effects. Examples of minimal lentiviralvectors are disclosed in WO-A-99/32646 and in WO-A-98/17815.

The absence of functional auxiliary genes from the retroviral vectorproduction system means that those functional genes will also be absentfrom retroviral vector particles produced by the system. Also, anyauxiliary proteins that would otherwise be encoded by those genes andincorporated into the vector particles will be absent from the vectorparticles. In known retroviral vector production systems, the auxiliarygenes may be present as part of the vector genome-encoding DNA, ortogether with the packaging components. The location of an auxiliarygene in a vector production system depends in part on its relationshipwith other retroviral components. For example, vif is often part of agag-pol packaging cassette in a packaging cell. Thus, to remove afunctional auxiliary gene for the purposes of the invention may involveits removal from the packaging components, or from the vector genome, orperhaps both.

To remove a functional auxiliary gene may not require removal of thegene in its entirety. Usually removal of part of the gene, or disruptionof the gene in some other way will be sufficient. The absence of afunctional auxiliary gene is understood herein to mean that the gene isnot present in a form in which it is capable of encoding the functionalauxiliary protein.

In a preferred system according to the invention, functional vpr and tatgenes or analogous genes normally present in the lentivirus on which thevector particles are based are both absent. These two auxiliary genesare associated with characteristics of lentiviruses that areparticularly undesirable for a gene therapy vector. However, other thanby the proviso given above, the invention is not limited with regard tothe combination of auxiliary genes that are absent in a system accordingto the invention for producing HIV-1-based vector particles, anycombination of three, or more preferably four, of the genes may beabsent in their functional form. Most preferably, all five of theauxiliary genes vpr, vif, tat, nef, and vpu are absent in theirfunctional form. Similarly, for systems concerned with otherlentiviruses, it is most preferable that all of the auxiliary genes areabsent in their functional form (except rev which is preferably presentunless replaced by a system analogous to the rev/RRE system).

Thus, preferably, the delivery system used in the invention is devoid ofat least tat and S2 (if it is an EIAV vector system), and possibly alsovif, vpr, vpx, vpu and nef. More preferably, the systems of the presentinvention are also devoid of rev. Rev was previously thought to beessential in some retroviral genomes for efficient virus production. Forexample, in the case of HIV, it was thought that rev and RRE sequenceshould be included. However, it has been found that the requirement forrev and RRE can be reduced or eliminated by codon optimisation (seebelow) or by replacement with other functional equivalent systems suchas the MPMV system. As expression of the codon-optimised gag-pol isrev-independent, RRE can be removed from the gag-pol expressioncassette, thus removing any potential for recombination with any RREcontained on the vector genome.

In a preferred embodiment, the viral genome of the present inventionlacks the Rev response element (RRE). In another preferred embodiment, anucleic acid sequence encoding Rev, or a functional equivalent thereof,is disrupted such that the nucleic acid sequence is incapable ofencoding the functional Rev or is removed from the vector genome.

In a preferred embodiment, the system used in the present invention isbased on a so-called “minimal” system in which some or all of theadditional genes have been removed. Preferably the viral vector of thepresent invention has a minimal viral genome.

As used herein, the term “minimal viral genome” means that the viralvector has been manipulated so as to remove the non-essential elementsand to retain the essential elements to provide the requiredfunctionality to infect, transduce and deliver a NOI to a target hostcell.

Preferably the viral vector with the minimal viral genome is a minimallentiviral vector.

Codon Optimisation

Codon optimisation has previously been described in WO99/41397.Different cells differ in their usage of particular codons. This codonbias corresponds to a bias in the relative abundance of particular tRNAsin the cell type. By altering the codons in the sequence to match withthe relative abundance of corresponding tRNAs, it is possible toincrease expression. By the same token, it is possible to decreaseexpression by deliberately choosing codons for which the correspondingtRNAs are known to be rare in the particular cell type. Thus, anadditional degree of translational control is available.

Many viruses, including HIV and other lentiviruses, use a large numberof rare codons and by changing these to correspond to commonly usedmammalian codons, increased expression of the packaging components inmammalian producer cells can be achieved. Codon usage tables are knownin the art for mammalian cells, as well as for a variety of otherorganisms.

Codon optimisation has a number of other advantages. By virtue ofalterations in their sequences, the nucleotide sequences encoding thepackaging components of the viral particles required for assembly ofviral particles in the producer cells/packaging cells have RNAinstability sequences (INS) eliminated from them. At the same time, theamino acid sequence coding sequence for the packaging components isretained so that the viral components encoded by the sequences remainthe same, or at least sufficiently similar that the function of thepackaging components is not compromised. Codon optimisation alsoovercomes the Rev/RRE requirement for export, rendering optimisedsequences Rev independent. Codon optimisation also reduces homologousrecombination between different constructs within the vector system (forexample, between the regions of overlap in the gag-pol and env openreading frames). The overall effect of codon optimisation is therefore anotable increase in viral titre and improved safety.

In one embodiment, only codons relating to INS are codon optimised.However, in a much more preferred and practical embodiment, thesequences are codon optimised in their entirety, with the exception ofthe sequence encompassing the frameshift site.

The gag-pol gene comprises two overlapping reading frames encoding gagand pol proteins respectively. The expression of both proteins dependson a frameshift during translation. This frameshift occurs as a resultof ribosome “slippage” during translation. This slippage is thought tobe caused at least in part by ribosome-stalling RNA secondarystructures. Such secondary structures exist downstream of the frameshiftsite in the gag-pol gene. For HIV, the region of overlap extends fromnucleotide 1222 downstream of the beginning of gag (wherein nucleotide 1is the A of the gag ATG) to the end of gag (nt 1503). Consequently, a281 bp fragment spanning the frameshift site and the overlapping regionof the two reading frames is preferably not codon optimised. Retainingthis fragment will enable more efficient expression of the gag-polproteins.

For EIAV, the beginning of the overlap has been taken to be nt 1262(where nucleotide 1 is the A of the gag ATG). The end of the overlap isat 1461 bp. To ensure that the frameshift site and the gag-pol overlapare preserved, the wild type sequence has been retained from nt 1156 to1465.

Derivations from optimal codon usage may be made, for example, toaccommodate convenient restriction sites, and conservative amino acidchanges may be introduced into the gag-pol proteins.

In a highly preferred embodiment, codon optimisation was based on highlyexpressed mammalian genes. The third and sometimes the second and thirdbase may be changed.

Due to the degenerate nature of the Genetic Code, it will be appreciatedthat a skilled worker can achieve numerous gag-pol sequences. Also,there are many retroviral variants described that can be used as astarting point for generating a codon optimised gag-pol sequence.Lentiviral genomes can be quite variable. For example, there are manyquasi-species of HIV-1 that are still functional. This is also the casefor EIAV. These variants may be used to enhance particular parts of thetransduction process. Details of HIV variants may also be found in theHIV databases maintained by Los Alamos National Laboratory. Details ofEIAV clones may be found at the NCBI database maintained by the NationalInstitutes of Health.

The strategy for codon optimised gag-pol sequences can be used inrelation to any retrovirus. This would apply to all lentiviruses,including EIAV, FIV, BIV, CAEV, VMR, SIV, HIV-1 and HIV-2. In addition,this method could be used to increase expression of genes from HTLV-1,HTLV-2, HFV, HSRV and human endogenous retroviruses (HERV), MLV andother retroviruses.

Codon optimisation can render gag-pol expression Rev independent. Toenable the use of anti-rev or RRE factors in the retroviral vector,however, it would be necessary to render the viral vector generationsystem totally Rev/RRE independent. Thus, the genome also needs to bemodified. This is achieved by optimising vector genome components.Advantageously, these modifications can also lead to the production of asafer system absent of all additional proteins both in the producer andin the transduced cell.

As described above, the packaging components for a retroviral vectorinclude expression products of gag, pol and env genes. In addition,efficient packaging depends on a short sequence of 4 stem loops followedby a partial sequence from gag and env (the “packaging signal”). Thus,inclusion of a deleted gag sequence in the retroviral vector genome (inaddition to the full gag sequence on the packaging construct) willoptimise vector titre. To date, efficient packaging has been reported torequire from 255 to 360 nucleotides of gag in vectors that still retainenv sequences, or about 40 nucleotides of gag in a particularcombination of splice donor mutation, gag and env deletions. It hassurprisingly been found that a deletion of all but the N-terminal 360nucleotides or so in gag leads to an increase in vector titre. Thus,preferably, the retroviral vector genome includes a gag sequence thatcomprises one or more deletions, more preferably the gag sequencecomprises about 360 nucleotides derivable from the N-terminus.

NOIs

In the present invention, the term NOI (nucleotide sequence of interest)includes any suitable nucleotide sequence, which need not necessarily bea complete naturally occurring DNA or RNA sequence. Thus, the NOI canbe, for example, a synthetic RNA/DNA sequence, a codon optimised RNA/DNAsequence, a recombinant RNA/DNA sequence (i.e. prepared by use ofrecombinant DNA techniques), a cDNA sequence or a partial genomic DNAsequence, including combinations thereof. The sequence need not be acoding region. If it is a coding region, it need not be an entire codingregion. In addition, the RNA/DNA sequence can be in a sense orientationor in an anti-sense orientation. Preferably, it is in a senseorientation. Preferably, the sequence is, comprises, or is transcribedfrom cDNA.

The NOI(s), also referred to as “heterologous sequence(s)”,“heterologous gene(s)” or “transgene(s)”, may be any one or more of, forexample, a selection gene(s), marker gene(s) and therapeutic gene(s).

The NOI may be a candidate gene that is of potential significance in adisease process. Thus the vector system of the present invention may,for example, be used for target validation purposes.

The NOI may have a therapeutic or diagnostic application. Suitable NOIsinclude, but are not limited to: sequences encoding enzymes, cytokines,chemokines, hormones, antibodies, anti-oxidant molecules, engineeredimmunoglobulin-like molecules, a single chain antibody, fusion proteins,immune co-stimulatory molecules, immunomodulatory molecules, anti-senseRNA, small interfering RNA (siRNA), a transdominant negative mutant of atarget protein, a toxin, a conditional toxin, an antigen, a tumoursuppresser protein and growth factors, membrane proteins, pro- andanti-angiogenic proteins and peptides, vasoactive proteins and peptides,anti-viral proteins and ribozymes, and derivatives thereof (such as withan associated reporter group). The NOIs may also encode pro-drugactivating enzymes. When used in a research context, the NOIs may alsoencode reporter genes such as, but not limited to, green fluorescentprotein (GFP), luciferase, β-galactosidase, or resistance genes toantibiotics such as, for example, ampicillin, neomycin, bleomycin,zeocin, chloramphenicol, hygromycin, kanamycin, among others.

The NOI may encode all or part of the protein of interest (“POI”), or amutant, homologue or variant thereof. For example, the NOI may encode afragment of the POI that is capable of functioning in vivo in ananalogous manner to the wild-type protein.

The term “mutant” includes POIs that include one or more amino acidvariations from the wild-type sequence. For example, a mutant maycomprise one or more amino acid additions, deletions or substitutions.

Here, the term “homologue” means an entity having a certain homologywith the NOI, or which encodes a protein having a degree of homologywith the POI. Here, the term “homology” can be equated with “identity”.

In the present context, a homologous sequence is taken to include anamino acid sequence that may be at least 75, 85 or 90% identical,preferably at least 95 or 98% identical to the subject sequence.Typically, the homologues will comprise the same active sites as thesubject amino acid sequence. Although homology can also be considered interms of similarity (i.e. amino acid residues having similar chemicalproperties/functions), in the context of the present invention, it ispreferred to express homology in terms of sequence identity.

In the present context, a homologous sequence is taken to include anucleotide sequence that may be at least 75, 85 or 90% identical,preferably at least 95 or 98% identical to the subject sequence.Typically, the homologues will comprise the same sequences that code forthe active sites etc. as the subject sequence. Although homology canalso be considered in terms of similarity (i.e. amino acid residueshaving similar chemical properties/functions), in the context of thepresent invention it is preferred to express homology in terms ofsequence identity.

Homology comparisons can be conducted by eye, or more usually, with theaid of readily available sequence comparison programs. Thesecommercially available computer programs can calculate percent (%)homology between two or more sequences.

A suitable computer program for carrying out sequence comparisons is theGCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereuxet al., 1984, Nucleic Acids Research 12:387). Examples of other softwarethan can perform sequence comparisons include, but are not limited to,the BLAST package (see Ausubel et al., 1999 ibid—Chapter 18), FASTA(Atschul et al., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suiteof comparison tools. Both BLAST and FASTA are available for offline andonline searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60).However, for some applications, it is preferred to use the GCG Bestfitprogram. The BLAST 2 Sequences tool is also available for comparingprotein and nucleotide sequence (see FEMS Microbiol Lett 1999 174(2):247-50; FEMS Microbiol Lett 1999 177(1): 187-8).

The sequences may also have deletions, insertions or substitutions ofamino acid residues that produce a silent change and result in afunctionally equivalent substance. Deliberate amino acid substitutionsmay be made on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues as long as the secondary binding activity of the substance isretained. For example, negatively charged amino acids include asparticacid and glutamic acid; positively charged amino acids include lysineand arginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine, valine,glycine, alanine, asparagine, glutamine, serine, threonine,phenylalanine, and tyrosine.

Conservative substitutions may be made, for example according to theTable below. Amino acids in the same block in the second column andpreferably in the same line in the third column may be substituted foreach other:

ALIPHATIC Non-polar G A P I L V Polar-uncharged C S T M N QPolar-charged D E K R AROMATIC H F W Y

The present invention also encompasses homologous substitution(substitution and replacement are both used herein to mean theinterchange of an existing amino acid residue, with an alternativeresidue) may occur i.e. like-for-like substitution such as basic forbasic, acidic for acidic, polar for polar etc. Non-homologoussubstitution may also occur i.e. from one class of residue to another.

Preferably the NOI encodes a single POI or a mutant, homologue orvariant thereof. In a highly preferred embodiment, the NOI does notencode a fusion protein. As used herein, the term “fusion protein” isused in its conventional sense to mean an entity that comprises two ormore protein activities, joined together by a peptide bond to form asingle chimeric protein. A fusion protein is encoded by a singlepolynucleotide driven by a single promoter.

Internal Ribosome Entry Site (IRES)

The viral genome of the present invention comprises at least one, butcan optionally comprise two or more NOIs. In order for two or more NOIsto be expressed, there may be two or more transcription units within thevector genome, one for each NOI. However, it is clear from theliterature that retroviral vectors achieve the highest titres and mostpotent gene expression properties if they are kept genetically simple(PCT/GB96/01230; Bowtell et al., 1988 J. Virol. 62, 2464; Correll etal., 1994 Blood 84, 1812; Emerman and Temin 1984 Cell 39, 459; Ghattaset al., 1991 Mol. Cell. Biol. 11, 5848; Hantzopoulos et al., 1989 PNAS86, 3519; Hatzoglou et al., 1991 J. Biol. Chem 266, 8416; Hatzoglou etal., 1988 J. Biol. Chem 263, 17798; Li et al., 1992 Hum. Gen. Ther. 3,381; McLachlin et al., 1993 Virol. 195, 1; Overell et al., 1988 Mol.Cell Biol. 8, 1803; Scharfman et al., 1991 PNAS 88, 4626; Vile et al.,1994 Gene Ther 1, 307; Xu et al., 1989 Virol. 171, 331; Yee et al., 1987PNAS 84, 5197). Thus, it is preferable to use an internal ribosome entrysite (IRES) to initiate translation of the second (and subsequent)coding sequence(s) in a poly-cistronic (or as used herein,“multicistronic”) message (Adam et al 1991 J. Virol. 65, 4985).

Insertion of IRES elements into retroviral vectors is compatible withthe retroviral replication cycle and allows expression of multiplecoding regions from a single promoter (Adam et al (as above); Koo et al(1992) Virology 186:669-675; Chen et al 1993 J. Virol 67:2142-2148).IRES elements were first found in the non-translated 5′ ends ofpicornaviruses where they promote cap-independent translation of viralproteins (Jang et al (1990) Enzyme 44: 292-309). When located betweenopen reading frames in an RNA, IRES elements allow efficient translationof the downstream open reading frame by promoting entry of the ribosomeat the IRES element followed by downstream initiation of translation.

As used herein, the term “cistron” refers to a nucleic acid segmentcorresponding to a polypeptide chain, comprising the relevanttranslational start (initiation) and stop (termination) codons. Amulticistronic mRNA is an mRNA transcript with more than one cistron andthus, encoding more than one polypeptide.

A review on IRES is presented by Mountford and Smith (TIG May 1995 vol11, No 5:179-184). A number of different IRES sequences are knownincluding those from encephalomyocarditis virus (EMCV) (Ghattas, I. R.,et al., Mol. Cell. Biol., 11:5848-5859 (1991); BiP protein [Macejak andSarnow, Nature 353:91 (1991)]; the Antennapedia gene of Drosophila(exons d and e) [Oh, et al., Genes & Development, 6:1643-1653 (1992)] aswell as those in poliovirus (PV) [Pelletier and Sonenberg, Nature 334:320-325 (1988); see also Mountford and Smith, TIG 11, 179-184 (1985)].

According to WO-A-97/14809, IRES sequences are typically found in the 5′non-coding region of genes. In addition to those in the literature theycan be found empirically by looking for genetic sequences that affectexpression and then determining whether that sequence affects the DNA(i.e. acts as a promoter or enhancer) or only the RNA (acts as an IRESsequence).

IRES elements from PV, EMCV and swine vesicular disease virus havepreviously been used in retroviral vectors (Coffin et al, as above).

The term “IRES” includes any sequence or combination of sequences whichwork as or improve the function of an IRES.

The IRES(s) may be of viral origin (such as EMCV IRES, PV IRES, or FMDV2A-like sequences) or cellular origin (such as FGF2 IRES, NRF IRES,Notch 2 IRES or EIF4 IRES).

For the IRES to be capable of initiating translation of each NOI, itshould be located between or prior to NOIs in the vector genome. Forexample, for a multicistronic sequence containing n NOIs, the genome maybe as follows:

-   -   [(NOI₁-IRES₁] . . . NOI_(n) n=1→n

For bi and tricistronic sequences, the order may be as follows:

-   -   NOI₁-IRES₁-NOI₂    -   NOI₁-IRES₁-NOI₂-IRES₂-NOI₃

Alternative configurations of IRESs and NOIs can also be utilised. Forexample transcripts containing the IRESs and NOIs need not be drivenfrom the same promoter.

An example of this arrangement may be:

-   -   IRES₁-NOI₁-promoter-NOI₂-IRES₂-NOI₃.

Preferably, in any construct utilising an internal cassette having morethan one IRES and NOI, the IRESs may be of different origins, that is,heterologous to one another. For example, one IRES may be from EMCV andthe other IRES may be from poliovirus.

Other Methods of Expressing Multiple Genes from One Vector

Although IRESs are an efficient way to co-express multiple genes fromone vector, other methods are also useful, and may be used alone or inconjunction with IRESs. These include the use of multiple internalpromoters in the vector (Overell et al., Mol Cell Biol. 8: 1803-8(1988)), or the use of alternate splicing patterns leading to multipleRNA species derived from the single viral genome that expresses thedifferent genes. This strategy has previously been used by itself fortwo genes (Cepko et al. Cell 37: 1053 (1984)).

Transduced Cells

The present invention also relates to a cell that has been transducedwith a vector system comprising a viral genome according to the firstaspect of the invention.

The cell may be transduced in vivo, in vitro or ex vivo. For example, ifthe cell is a cell from a mammalian subject, the cell may be removedfrom the subject and transduced ready for reimplantation into thesubject (ex vivo transduction). Alternatively, the cell may betransduced by direct gene transfer in vivo, using the vector system ofthe present invention in accordance with standard techniques (such asvia injection of vector stocks expressing the NOIs). If the cell is partof a cell line that is stable in culture (i.e. which can survivenumerous passages and can multiple in vitro) then it may be transducedin vitro by standard techniques, for example, by exposure of the cell toviral supernatants comprising vectors expressing the NOIs.

The cell may be any cell that is susceptible to transduction. If thevector system is capable of transducing non-dividing cells (for exampleif it is a lentiviral system) then the cell may be a non-dividing cell,such as a neuron.

Cassettes

The present invention can employ cassettes comprising one or more NOIs,which, in the case of two or more NOIs, can be operably linked by anIRES. These cassettes may be used in a method for producing the vectorgenome in a producer cell.

The present invention also provides an expression vector comprising sucha cassette. Transfection of a suitable cell with such an expressionvector should result in a cell that expresses each POI encoded by theNOI in the cassette. The present invention also provides such atransfected cell.

Cloning of the cassette into an expression vector and transfection ofcells with the vector (to give expression of the cassette) can becarried out by techniques well known in the art (such as those describedin Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition,Cold Spring Harbor Laboratory press (1989)), and other laboratorytextbooks).

Preferably the cassette comprises a promoter. A cassette comprising twoor more NOIs can be bicistronic or tricistronic, and can comprises thefollowing elements:

Promoter-(NOI₁)-(IRES₁)-(NOI₂)Promoter-(NOI₁)-(IRES₁)-(NOI₂)-(IRES₂)-(NOI₃) PharmaceuticalCompositions

The present invention provides a pharmaceutical composition, whichcomprises a vector genome according to the second aspect of theinvention and a pharmaceutically acceptable carrier, diluent orexcipient (including combinations thereof).

The pharmaceutical compositions may be for human or animal usage inhuman and veterinary medicine or research, and will typically compriseany one or more of a pharmaceutically acceptable diluent, carrier, orexcipient. Acceptable carriers or diluents for therapeutic use are wellknown in the pharmaceutical art, and are described, for example, inRemington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaroedit. 1985). The choice of pharmaceutical carrier, excipient or diluentcan be selected with regard to the intended route of administration andstandard pharmaceutical practice. The pharmaceutical compositions maycomprise as—or in addition to—the carrier, excipient or diluent anysuitable binder(s), lubricant(s), suspending agent(s), coating agent(s),solubilising agent(s).

Preservatives, stabilizers, dyes and even flavouring agents may beprovided in the pharmaceutical composition. Examples of preservativesinclude sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid. Antioxidants and suspending agents may be also used.

There may be different composition/formulation requirements dependent onthe different delivery systems. By way of example, the pharmaceuticalcomposition of the present invention may be formulated to be deliveredusing a mini-pump or by a mucosal route, for example, as a nasal sprayor aerosol for inhalation or ingestible solution, or parenterally inwhich the composition is formulated by an injectable form, for delivery,by, for example, an intravenous, intramuscular or subcutaneous route.Alternatively, the formulation may be designed to be delivered by bothroutes.

Where the pharmaceutical composition is to be delivered mucosallythrough the gastrointestinal mucosa, it should be able to remain stableduring transit though the gastrointestinal tract; for example, it shouldbe resistant to proteolytic degradation, stable at acid pH and resistantto the detergent effects of bile.

Where appropriate, the pharmaceutical compositions can be administeredby inhalation, in the form of a suppository or pessary, topically in theform of a lotion, solution, cream, ointment or dusting powder, by use ofa skin patch, orally in the form of tablets containing excipients suchas starch or lactose or chalk, or in capsules or ovules either alone orin admixture with excipients, or in the form of elixirs, solutions orsuspensions containing flavouring or colouring agents, or they can beinjected parenterally, for example, intravenously, intramuscularly orsubcutaneously. For parenteral administration, the compositions may bebest used in the form of a sterile aqueous solution, which may containother substances, for example, enough salts or monosaccharides to makethe solution isotonic with blood. For buccal or sublingualadministration, the compositions may be administered in the form oftablets or lozenges that can be formulated in a conventional manner.

Administration

Typically, a physician will determine the actual dosage that will bemost suitable for an individual subject and it will vary with the age,weight and response of the particular patient and severity of thecondition. The dosages below are exemplary of the average case. Therecan, of course, be individual instances where higher or lower dosageranges are merited.

The compositions (or component parts thereof) of the present inventionmay be administered orally. In addition, or in the alternative, thecompositions (or component parts thereof) of the present invention maybe administered by direct injection. In addition, or in the alternative,the compositions (or component parts thereof) of the present inventionmay be administered topically. In addition, or in the alternative, thecompositions (or component parts thereof) of the present invention maybe administered by inhalation. In addition, or in the alternative, thecompositions (or component parts thereof) of the present invention mayalso be administered by one or more of: parenteral, mucosal,intramuscular, intravenous, subcutaneous, intraocular or transdermaladministration means, and are formulated for such administration.

By way of further example, the pharmaceutical composition of the presentinvention may be administered in accordance with a regimen of 1 to 10times per day, such as once or twice per day. The specific dose leveland frequency of dosage for any particular patient may be varied andwill depend upon a variety of factors including the activity of thespecific compound employed, the metabolic stability and length of actionof that compound, the age, body weight, general health, sex, diet, modeand time of administration, rate of excretion, drug combination, theseverity of the particular condition, and the host undergoing therapy.

The term “administered” also includes, but is not limited to, deliveryby a mucosal route, for example, as a nasal spray or aerosol forinhalation or as an ingestible solution; a parenteral route wheredelivery is by an injectable form, such as, for example, an intravenous,intramuscular or subcutaneous route.

Hence, one or more of the following routes may administer thepharmaceutical composition of the present invention: oraladministration, injection (such as direct injection), topical,inhalation, parenteral administration, mucosal administration,intramuscular administration, intravenous administration, subcutaneousadministration, intraocular administration or transdermaladministration.

Diseases

Pharmaceutical compositions comprising an effective amount of vectorcomprising an identified modulating moiety operably linked to an NOI maybe used in the treatment of disorders, such as those listed inWO-A-98/09985. For ease of reference, part of that list is now provided:macrophage inhibitory and/or T cell inhibitory activity and thus,anti-inflammatory activity; anti-immune activity, i.e. inhibitoryeffects against a cellular and/or humoral immune response, including aresponse not associated with inflammation; diseases associated withviruses and/or other intracellular pathogens; inhibit the ability ofmacrophages and T cells to adhere to extracellular matrix components andfibronectin, as well as up-regulated fas receptor expression in T cells;inhibit unwanted immune reaction and inflammation including arthritis,including rheumatoid arthritis, inflammation associated withhypersensitivity, allergic reactions, asthma, systemic lupuserythematosus, collagen diseases and other autoimmune diseases,inflammation associated with atherosclerosis, arteriosclerosis,atherosclerotic heart disease, reperfusion injury, cardiac arrest,myocardial infarction, vascular inflammatory disorders, respiratorydistress syndrome or other cardiopulmonary diseases, inflammationassociated with peptic ulcer, ulcerative colitis and other diseases ofthe gastrointestinal tract, hepatic fibrosis, liver cirrhosis or otherhepatic diseases, thyroiditis or other glandular diseases,glomerulonephritis or other renal and urologic diseases, otitis or otheroto-rhino-laryngological diseases, dermatitis or other dermal diseases,periodontal diseases or other dental diseases, orchitis orepididimo-orchitis, infertility, orchidal trauma or other immune-relatedtesticular diseases, placental dysfunction, placental insufficiency,habitual abortion, eclampsia, pre-eclampsia and other immune and/orinflammatory-related gynecological diseases, posterior uveitis,intermediate uveitis, anterior uveitis, conjunctivitis, chorioretinitis,uveoretinitis, optic neuritis, intraocular inflammation, e.g. retinitisor cystoid macular oedema, sympathetic ophthalmia, scleritis, retinitispigmentosa, immune and inflammatory components of degenerative fondusdisease, inflammatory components of ocular trauma, ocular inflammationcaused by infection, proliferative vitreo-retinopathies, acute ischaemicoptic neuropathy, excessive scarring, e.g. following glaucoma filtrationoperation, immune and/or inflammation reaction against ocular implantsand other immune and inflammatory-related ophthalmic diseases,inflammation associated with autoimmune diseases or conditions ordisorders where, both in the central nervous system (CNS) or in anyother organ, immune and/or inflammation suppression would be beneficial,Parkinson's disease, complication and/or side effects from treatment ofParkinson's disease, AIDS-related dementia complex HIV-relatedencephalopathy, Devic's disease, Sydenham chorea, Alzheimer's diseaseand other degenerative diseases, conditions or disorders of the CNS,inflammatory components of stokes, post-polio syndrome, immune andinflammatory components of psychiatric disorders, myelitis,encephalitis, subacute sclerosing pan-encephalitis, encephalomyelitis,acute neuropathy, subacute neuropathy, chronic neuropathy,Guillain-Barre syndrome, Sydenham chora, myasthenia gravis,pseudo-tumour cerebri, Down's Syndrome, Huntington's disease,amyotrophic lateral sclerosis, inflammatory components of CNScompression or CNS trauma or infections of the CNS, inflammatorycomponents of muscular atrophies and dystrophies, and immune andinflammatory related diseases, conditions or disorders of the centraland peripheral nervous systems, post-traumatic inflammation, septicshock, infectious diseases, inflammatory complications or side effectsof surgery, bone marrow transplantation or other transplantationcomplications and/or side effects, inflammatory and/or immunecomplications and side effects of gene therapy, e.g. due to infectionwith a viral carrier, or inflammation associated with AIDS, to suppressor inhibit a humoral and/or cellular immune response, to treat orameliorate monocyte or leukocyte proliferative diseases, e.g. leukaemia,by reducing the amount of monocytes or lymphocytes, for the preventionand/or treatment of graft rejection in cases of transplantation ofnatural or artificial cells, tissue and organs such as cornea, bonemarrow, organs, lenses, pacemakers, natural or artificial skin tissue.Specific cancer related disorders include but not limited to: solidtumours; blood born tumours such as leukemias; tumor metastases; benigntumours, for example hemangiomas, acoustic neuromas, neurofibromas,trachomas, and pyogenic granulomas; rheumatoid arthritis; psoriasis;ocular angiogenic diseases, for example, diabetic retinopathy,retinopathy of prematurity, macular degeneration, corneal graftrejection, neovascular glaucoma, retrolental fibroplasia, rubeosis;Osler-Webber Syndrome; myocardial angiogenesis; plaqueneovascularization; telangiectasia; hemophiliac joints; angiofibroma;wound granulation; coronary collaterals; cerebral collaterals;arteriovenous malformations; ischaemic limb angiogenesis; neovascularglaucoma; retrolental fibroplasia; diabetic neovascularization;Helicobacter-related diseases, fractures, vasculogenesis, hematopoiesis,ovulation, menstruation and placentation.

Various preferred features and embodiments of the present invention willnow be described in more detail by way of non-limiting examples.

EXAMPLES Example 1 Alterations to the Woodchuck Hepatitis VirusPost-Transcriptional Regulatory Element (WPRE) to Increase the SafetyProfile of Viral Vectors

A study was conducted to evaluate administration of our EIAV vectors viathe main artery to foetal mice in utero. After birth, the mice weremonitored for long periods. Mice transduced with an EIAV vectorcontaining the wild-type WPRE (pSMART2 and 3) developed liver tumourswhile vectors that did not contain the WPRE (pONY8 series) did not. Thetumours were associated only with the liver, and were not observed inother organs. Use of the pSMART2Z vector led to development of livertumours within 3 months of birth. The results indicate that the presenceof a WPRE with the wild-type nucleotide sequence often resulted in thedevelopment of liver tumours following in utero administration ofvector. This may be due to effects resulting from partial functioning ofthe X promoter and truncated X-polypeptide. This is a surprisingobservation given that the X-protein promoter requires WHV proteins tofunction fully. Insertion of the WPRE in retroviral or lentiviralvectors in the reverse orientation leads to a reduction in proteinexpression, as measured by marker gene expression studies (Zufferey, R.,et al, (1999) J. Virol. 73: 2886-92). The mechanism for this inhibitionmay be due to antisense effects resulting from transcription from the Xpromoter (Zufferey, R., et al, ibid). Therefore, it is possible thatlow-level transcription from the X promoter leads to accumulation of atruncated X protein polypeptide that contributes to the formation ofliver tumours.

Given this observation, the ability of the WPRE to express the X-proteinwould need to be abrogated. To achieve this, mutation of nucleotideswithin the X promoter region, or within the translation initiation codon(ATG) of the X protein itself, or preferably both could be introduced.Maximal diversity from the wild type WPRE sequence would be thepreferred option, as this will prevent reversion to wild-type sequencefrom occurring.

Given that the retroviral reverse transcriptase (in this caseoriginating from the lentivirus, EIAV) has a low fidelity (an averagesingle mistake incorporated per 10,000 nucleotides reverse transcribed),then the likelihood of a single base pair change reverting an alterednucleotide back to wild type will occur at a frequency of 1 per 40,000integration events. Therefore, with each change relative to thewild-type WPRE sequence comes an additive reduction in the likelyreversion to functional wild-type sequence. However, balanced with thedesire to maximise diversity from the wild-type sequence is the need toretain WPRE functionality: there are presumably a limited number ofchanges that can be made to the WPRE without affecting its ability toenhance transgene expression.

A set of nucleotide changes within the WPRE that maintain its positiveeffects on transgene expression is provided, while dramatically loweringthe probability of reversion to wild type. This altered WPRE sequence isreferred to as WPREMut (SEQ ID NO:1).

The mutations were inserted by overlap PCR using the following primers:

1. 5′-GTG AAT TCG CGG CCG CAA TCA (SEQ ID NO:8) ACC TCT 2. 5′-GGT GGCAAC ACA GGC GAG CAG (SEQ ID NO:9) CCC CGA GTC TCA GCA GAC CTT CCC CGACAA C 3. 5′-CCG TGG TGT TGT CGG GGA AGG (SEQ ID NO:10) TCT GCT GAG ACTCGG 4. 5′-GCT GTC GAG CGG CCG CGA ATT (SEQ ID NO:11) CAC TAG TGA TTC TCGAC

Primers 1 and 2 were used to amplify a 452 bp PCR product using thewild-type WPRE sequence as template (PCR#1). Primers 3 and 4 were usedto amplify a 260 bp PCR product using the wild-type WPRE sequence astemplate (PCR#2). PCR#1 and PCR#2 were used in overlap PCR to createWPREMut (652 bp). WPREMut was inserted into vector genomes using theflanking Not I sites.

A comparison of the expression of a reporter gene (eGFP) from pSMART3Gvectors containing either wild-type WPRE, or WPREMut was conducted.Transductions of D17 cells were conducted using dilutions of the vectorpreps and pooled transduced cells were analysed for eGFP expression byFACS from each dilution. Samples containing between 1% and 10%GFP-positive cells were used in order to compare the expression level ofGFP, in order to control for differences in vector titre. The meanfluorescence intensity (MFI) of eGFP expression for each of thesesamples was used; the average MFI±SD is shown in FIG. 5.

The sum of the 13 changes within the WPREMut relative to wild-type WPREis predicted to reduce the probability of reversion to wild-typesequence to a very small level. FIG. 6 shows an alignment of WPREsequences showing the mutations that have been introduced into the WPRE.

To study the effects of using the WPRE with introduced changes (WPREMut)in a relevant system, a study will compare vectors containing thewild-type WPRE and those containing the mutated WPRE. To achieve this,the WPRE in pSMART2Z has been swapped with the WPREMut sequence tocreate pSMART2ZWPREMut. These two vectors were made in parallel usingthe same production process (co-transfection of HEK293T cells withpONY3.1 (wild-type Gag/Pol expression construct) and pRV67 (VSV-Gexpression construct). In addition to these two vectors, a latestgeneration EIAV vector genome will be tested in parallel (pONYT9.1NCZ).This vector genome differs from pSMART2Z in a number of ways (inaddition to the WPREMut sequence): i) a tetracycline resistance genereplacing the ampicillin resistance gene in earlier vector genomes (eg.pSMART2Z); ii) mutations within the major splice donor site to preventunwanted splicing; iii) mutations within the initiation codon of thefirst exon of EIAV Tat (CTG) to prevent translation of the truncated Tatpolypeptide; iv) mutations of all the ATG sequences within the packagingsignal to ATTG (Ψ; pSMART2Z vectors contained the first two ATGsequences mutated in this fashion); v) a neomycin transferase genedownstream of the Ψ site that allows production of high titre vector inthe absence of Rev; vi) minimal retained nucleotide sequencecorresponding to a small region of EIAV env (pSMART2 vectors contain alarger fragment from this region of EIAV). The pONYT9.1NCZ vector ismade by transient transfection of HEK293T cells with pESGPK (the codonoptimised Gag/Pol expression construct, with a kanamycin resistancegene) and pRVK (a kanamycin resistant variant of pRV67, the VSV-Gexpression construct). The vectors to be compared in this study aresummarised in Table 1.

Mice will be treated as for the previous study (administration of testarticle via the main artery to foetal mice in utero) and observed forextended periods for the generation of tumours. The results of thisstudy will demonstrate whether the wild-type WPRE sequence is indeedresponsible for the generation of tumours in this model. These resultswill provide evidence supporting the use of vectors containing theimproved WPRE (WPREMut) rather than wild-type WPRE in vivo.

TABLE 1 Vectors to be used in comparative study of EIAV vectors in themurine in utero transduction model. Gag/Pol VSV-G Test expressionexpression WPRE article Vector genome cassette cassette (WT/Mut) 1pSMART2Z pONY3.1 pRV67 WT 2 pSMART2Z WPREMut pONY3.1 pRV67 Mut 3pONYT9.1NCZ pESGPK pRVK Mut 4 Formulation buffer N/A N/A N/A

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inchemistry, biology or related fields are intended to be within the scopeof the following claims.

1. An isolated nucleic acid molecule comprising a woodchuckpost-transcriptional regulatory element (WPRE) containing an X region,wherein the WPRE has a mutation in the X region whereby expression of afunctional X protein is prevented.
 2. The isolated nucleic acid moleculeof claim 1, wherein the mutation in the X region further preventsreversion to wild type WPRE.
 3. The isolated nucleic acid molecule ofclaim 1, comprising the sequence of SEQ ID NO:1.
 4. The isolated nucleicacid molecule of claim 1, wherein the X region comprises a promotersequence and wherein the mutation is in the promoter sequence.
 5. Theisolated nucleic acid molecule of claim 1, wherein the X regioncomprises an initiation codon and wherein the mutation is in theinitiation codon.
 6. The isolated nucleic acid molecule of claim 1,wherein the X protein is not expressed.
 7. The isolated nucleic acidmolecule of claim 1, wherein the X protein is non-functional.
 8. Aretroviral vector genome comprising at least one NOI and the isolatednucleic acid molecule of claim
 1. 9. The retroviral vector genome ofclaim 8, which is a lentiviral vector genome.
 10. The retroviral vectorgenome of claim 8, wherein the retroviral vector genome comprises aself-inactivating (SIN) LTR.
 11. The retroviral vector genome of claim9, wherein the lentiviral vector genome is a minimal lentiviral vectorgenome.
 12. The retroviral vector genome according to claim 9, wherein anucleic acid sequence encoding Rev, or a functional equivalent thereof,is disrupted such that the nucleic acid sequence is incapable ofencoding the functional Rev or is removed from the vector genome. 13.The retroviral vector genome according to claim 9, wherein a nucleicacid sequence encoding Tat is disrupted such that the nucleic acidsequence is incapable of encoding functional Tat or is removed from thevector genome.
 14. The retroviral vector genome of claim 9, wherein thelentiviral vector genome is derived from a viral species selected fromthe group consisting of human immunodeficiency virus (HIV), simianimmunodeficiency virus (SIV), visna/maedi virus (VMV), caprinearthritis-encephalitis virus (CAEV), equine infectious anaemia virus(EIAV), feline immunodeficiency virus (FIV) and bovine immunodeficiencyvirus (BIV).
 15. The retroviral vector genome of claim 9, wherein thelentiviral vector genome is derived from a non-primate lentivirus. 16.The retroviral vector genome of claim 9, wherein the retroviral vectorgenome comprises a central polypurine tract (cPPT) sequence.
 17. Theretroviral vector genome of claim 9, wherein the retroviral vectorgenome comprises a gag packaging signal having ATG motifs, and whereinthe ATG motifs are ATTG motifs.
 18. The retroviral vector genome ofclaim 8, wherein the retroviral vector genome is multicistronic.
 19. Theretroviral vector genome of claim 18, wherein the retroviral vectorgenome comprises at least one internal regulatory element.
 20. Theretroviral vector genome of claim 19, wherein the internal regulatoryelement is a promoter or an internal ribosomal entry site (IRES).
 21. Aretroviral vector system for producing a retrovirus-derived vectorparticle, comprising: (i) the retroviral vector genome of claim 8; (ii)a nucleotide sequence encoding retroviral gag and pol proteins; and(iii) nucleotide sequences encoding other essential viral packagingcomponents not encoded by the nucleotide sequence of ii).
 22. Theretroviral vector system of claim 21, wherein nucleic acid sequence(s)encoding at least one of Vpr, Vif, Tat, Nef, or analogous auxiliarygenes, from the retrovirus from which the particles are derived, aredisrupted such as said nucleic acid sequence(s) are incapable ofencoding functional Vpr, Vif, Tat, Nef, or analogous auxiliary proteins,or are removed from the system.
 23. The retroviral vector system ofclaim 21, wherein the vector system is pseudotyped with at least part ofa heterologous env protein.
 24. The retroviral vector system of claim23, wherein the heterologous env protein is derivable from Rabies-G orVSV-G.
 25. A retroviral particle produced from the retroviral vectorsystem of claim
 21. 26. A cell that has been transduced with theretroviral vector system of claim
 21. 27. A composition comprising theretroviral vector genome of claim 8, together with a carrier or diluent.28. A composition comprising the viral particle of claim 25, togetherwith a carrier or diluent.
 29. A method of delivering at least one NOIto a target cell, comprising introducing the retroviral vector genome ofclaim 8 into the target cell, whereby the NOI is delivered to the targetcell.
 30. A retroviral vector comprising at least one NOI and a nucleicacid molecule comprising a woodchuck post-transcriptional regulatoryelement (WPRE) containing an X region, wherein the WPRE has a mutationin the X region whereby expression of a functional X protein isprevented.
 31. The retroviral vector of claim 30, wherein the mutationin the X region further prevents reversion to wildtype WPRE.
 32. Amethod of making a retroviral particle comprising the steps of: (i)introducing the retroviral vector of claim 30 into a packaging cell, orintroducing the retroviral vector of claim 30 together with nucleic acidsequence(s) encoding gag/pol and envelope proteins into a producer cell;and (ii) obtaining the retroviral vector particle therefrom, whereinsaid retroviral vector particle comprises the at least one NOI and thenucleic acid molecule comprising the mutated WPRE.
 33. The method ofclaim 32, wherein the envelope is a heterologous envelope proteinselected from the group consisting of Rabies G and VSV-G.
 34. A methodof delivering at least one NOI to a target cell, comprising introducingthe retroviral vector of claim 30 into the target cell, whereby the NOIis delivered to the target cell.
 35. A method of identifying a geneinvolved in tumorigenesis, said method comprising the steps of: (i)introducing the isolated nucleic acid of claim 1 into a cell ofinterest, whereby the nucleic acid is recombined into chromosomal DNA ofthe cell of interest; (ii) determining whether the cell of interestforms a tumor; and, if the cell of interest forms a tumor; (iii)locating a site of recombination in the chromosomal DNA; and (iv)identifying a gene near or adjacent to the site of recombination;thereby identifying the gene involved in tumorigenesis.